Prolonged release of GM-CSF

ABSTRACT

Formulations for controlled, prolonged release of GM-CSF have been developed. These are based on solid microparticles formed of the combination of biodegradable, synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and copolymers thereof with excipients and drug loadings that yield zero order or first order release, or multiphasic release over a period of approximately three to twenty one days, preferably one week, when administered by injection. In the preferred embodiment, the microparticles are microspheres having diameters in the range of 10 to 60 microns, formed of a blend of PLGA having different molecular weights, most preferably 6,000, 30,000 and 41,000. Other embodiments have been developed to alter the release kinetics or the manner in which the drug is distributed in vivo. For example, in some cases a polymer is selected which elicits a mild inflammatory reaction, for example, PLGA and polyanhydrides can act as chemoattractant, either due to the polymer itself or minor contaminants in the polymer, or polymers which are bioadhesive are used for transmucosal or oral delivery. In another embodiment, the GM-CSF is administered in a hydrogel which can be injected subcutaneous or at a specific site for controlled release. The microparticles or hydrogel are administered to the patient in an amount effect to stimulate proliferation of hematopoietic cells, especially white cells.

This is a continuation of U.S. Ser. No. 09/185,213, filed Nov. 3, 1998,now U.S. Pat. No. 6,120,897 which is a divisional of U.S. Ser. No.08/542,445, filed Oct. 12, 1995, now U.S. Pat. No. 5,942,253.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of controlled, prolongedrelease microsphere formulations for recombinant human granulocytemacrophage colony stimulating factor (GM-CSF).

GM-CSF, granulocyte macrophage colony stimulating factor, is ahematopoietic growth factor which promotes the proliferation anddifferentiation of hematopoietic progenitor cells. The cloned gene forGM-CSF has been expressed in bacteria, yeast and mammalian cells. Theendogenous human protein is a monomeric glycoprotein with a molecularweight of about 22,000 daltons. GM-CSF produced in a yeast expressionsystem is commercially available as Leukine® from Immunex Corporation,Seattle, Washington. It is a glycoprotein of 127 amino acidscharacterized by three primary molecular species having molecular massesof 19,500, 16,800, and 15,500 daltons.

Generally, GM-CSF is administered over a period of at least 6 to 7 daysin order to obtain the optimal effect on the white blood cells. Undersome circumstances, it is desirable to have a formulation which providescontinuous, zero order or first order kinetic release of GM-CSF over aperiod of approximately one week. Moreover, sustained releaseformulation of GM-CSF may have advantageous therapeutic utilities notshared by standard liquid formulations. Sustained-release formulationsof GM-CSF, however, are not currently available.

Controlled release formulations are well known for drug delivery. Bothbiodegradable and non-biodegradable polymers have been used to formmicrocapsules, microspheres or microparticles of various diameters,porosities, and drug loadings with the goal of obtaining release of theencapsulated drug over a period of time, Many formulations that havebeen developed have been designed for administration by injection,although the majority of controlled release formulations have entericcoatings or are formulations resistant to passage through thegastrointestinal tract that have been developed for oral administration.

It is difficult to achieve linear, controlled release using the standardformulations. Most formulations are designed either to provide veryrapid release by diffusion and/or degradation of the polymer forming themicroparticle or provide for a burst release followed by some kind oflinear release which generally plateaus after a period of tine. U.S.Pat. No. 5,192,741 to Orsolini, et al., is representative of theliterature regarding the difficulties in obtaining controlled releasefrom microspheres formed of poly(lactide-co-glycolides) (PL3As).Similarly, lu and Park J. Pharm. Sci. Technical 49, 13-19 (1995)describes the use of microcapsules, noting that one cannot obtain goodrelease characteristics with microspheres and that protein stability inthe microspheres is a problem. Since GM-CSF is an extremely potentcompound where the effect may vary widely depending upon the givendosage, it may be advantageous in some circumstances to obtain a morelinear release rather than a burst followed by a plateau of drug beingreleased.

Representative of the many patents relating to controlled release areU.S. Pat. No. 4,767,628 to Hutchinson, disclosing multiphasic release ofa peptide from a PLGA carrier. Blends of polymers are used is a largematrix delivery system to avoid multiphasic release. U.S. Pat. No.4,897,268 to Tice, et al., discloses the use of different PLGAs in thesame composition, but blends microspheres made of the different PLGAs toachieve linear release. U.S. Pat. No. 4,849,228 to Yamamoto, et al.,claims PLGA microspheres having a very low monobasic acid content whichallegedly have excellent release characteristics.

It is therefore an object of the present invention to provide aformulation encapsulating GM-CSF which provides for controlled,prolonged release with either zero order kinetics, first order releasekinetics or multiphasic release kinetics over a period of greater thanone day following administration to a patient by injection.

It is a further object of the present invention to provide a formulationfor delivery of GM-CSF for administration orally, transmucosally,topically or by injection.

SUMMARY OF THE INVENTION

Formulations for controlled, prolonged release of GM-CSF have beendeveloped. These are based on solid microparticles formed of thecombination of biodegradable, synthetic polymers such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), and copolymers thereof withexcipients and drug loadings that yield a sustained release over aperiod of one day to at least one week, when administered orally,transmucosally, topically or by injection. In the preferred embodiment,the microparticles have different as diameters depending on their routeof administration. Microparticles administered by injection havediameters sufficiently small to pass through a needle, in a size rangeof between 10 and 100 microns. Orally administered microparticles areless than 10 microns in diameter to facilitate uptake by the Peyer'spatches in the small intestine.

Other embodiments have been developed to alter the release kinetics orthe manner in which the drug is distributed in vivo. For example, insome cases a polymer is selected which elicits a mild inflammatoryreaction, for example, PLGA and polyanhydrides, which can act aschemoattractant, either due to the polymer itself or minor contaminantsin the polymer. In another embodiment, the GM-CSF is administered in ahydrogel which can be injected subcutaneous or at a specific site forcontrolled release.

The microparticles or hydrogel are administered to the patient in anamount effective to stimulate proliferation of hematopoietic cells,especially white cells. These are most preferably microspheresadministered by injection.

Examples demonstrate the preparation of microparticles releasing GM-CSFover a prolonged period with zero order, first order, or multiphasicrelease kinetics. The type of release kinetics are determined for theparticular clinical application. The data demonstrates that it ispossible not only to achieve the desired release characteristics butalso to retain extremely high levels of bioactivity of the encapsulatedGM-CSF. Examples also demonstrate release from hydrogels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of GM-CSF release, mean percent cumulative release invitro over time (days) for a 1% load (squares) and 3% load (diamonds) inmicrospheres prepared by phase separation with a single PLGA copolymer.

FIG. 1B is a graph of GM-CSF release, mean percent cumulative release invitro over time (days) for a 1.54% load (squares, lot B4), 1.28% load(diamonds, lot O4) and 1.5% load (circles, lot V4) in microspheresprepared by phase separation using a blend of PLA and PLGA polymers.

FIG. 2A is a graph of in vitro release kinetics for “Lot O” microspheresprepared by phase separation of a single molecular weight PLGA, showingGM-CSF release as percent cumulative release in vitro over time (days).

FIG. 2B is a graph of mouse serum GM-CSF levels (ng/ml) over time (days)following microsphere or bolus injections, for 50 mg microspheres, 500μg bolus, and 50 μg bolus.

FIG. 2C is a graph of the GM-CSF levels following microsphere injection(diamonds) versus levels calculated from in vitro release rate andexperimental half-life (line)

FIG. 3A is a graph of GM-CSF release, mean percent cumulative release invitro over time (days) for lot V4 microspheres prepared by phaseseparation using a blend of two PLGAs of different molecular weight anda PLA.

FIG. 3B is a graph of TF-1 bioactivity of lot V4 release samples,percent activity at discrete time points (days).

FIG. 3C are graphs of the white blood cell counts (WBC), absoluteneutrophil counts (ANC), and platelet counts in primates injected withmicrospheres containing GM-CSF, as a function of time (days).

FIG. 4 is a graph of GM-CSF release from a PLGA gel, percent cumulativerelease over time (days).

FIG. 5 is a graph of TF-1 cell activity of GM-CSF extracted from PLGAmicrospheres with acetic acid, graphing percent activity versusmicrosphere lot.

FIG. 6 is a graph of PLGA degradation over time of three types ofmicrospheres prepared from either PLGA (Cytec. 7 I.V.), PLA (R104) or an80/20 blend of the two polymers, graphing weight average molecularweight over time (days).

DETAILED DESCRIPTION OF THE INVENTION

There are many advantages for a controlled release formulation ofGM-CSF. Among these are the convenience of a single injection for thepatient and physician, avoidance of peaks and valleys in systemic GM-CSFconcentration which is associated with repeated injections, thepotential to reduce the overall dosage of GM-CSF, and the potential toenhance the pharmacological effects of GM-CSF. A controlled releaseformulation of GM-CSF also provides an opportunity to use GM-CSF in amanner not previously exploited, such as a vaccine adjuvant.

Controlled Release Formulations

As used herein, “sustained” or “extended” release of the GM-CSF can becontinuous or discontinuous, linear or non-linear. This can beaccomplished using one or more types of polymer compositions, drugloadings, inclusion of excipients or degradation enhancers, or othermodifiers, administered alone, in combination or sequentially to producethe desired effect. Zero order or linear release is generally construedto mean that the amount of GM-CSF released over time remains relativelyconstant as a function of amount/unit time during the desired timeframe, for example, six to seven days. Multi-phasic is generallyconstrued to mean that release occurs in more than one “burst”.

As used herein, “microparticles” refers to particles having a diameterof less than one mm, more typically less than 100 microns. Micropaticlescan refer to microspheres, which are solid spherical microparticles, andmicrocapsules, which are spherical microparticles having a core of adifferent polymer, drug, or composition. Unless otherwise stated herein,microparticles refers to solid particles, not microcapsules.

Polymers for Formation of Microparticles

Many polymers have been used for controlled drug delivery.

Polymers typically are thermoplastic synthetic polymers, such asethylenevinyl acetate and poly(acrylic acid), which are generally viewedas non-biodegradable since they remain in relatively the same form overa period of at least two or tree years following implantation in thebody, and biodegradable polymers, such as poly(hydroxy acids) includingpolylactic acid, polyglycolic acid, and copolymers thereof,polyanhydrides, polyorthoesters, and certain types of protein andpolysaccharide polymers. The term bioerodible or biodegrable, as usedherein, means a polymer that dissolves or degrades within a period thatis acceptable in the desired application (usually in vivo therapy), lessthan about five years and most preferably less than about one year, onceexposed to a physiological solution of pH 6-8 at a temperature ofbetween about 25° C. and 38° C.

A preferred polymer material is one which is biodegradable and whichretains sufficient form to control release for a period followingimplantation of at least six to seven days. The poly (hydroxy acids),especially poly(lactic acid-co-glycolic acid) (“PLGA”), is aparticularly preferred polymer since it has been used in the manufactureof degradable sutures for several decades. The polymer degrades byhydrolysis following exposure to the aqueous environment of the body.The polymer is hydrolyzed to yield lactic and glycolic acid monomers,which are normal byproducts of cellular metabolism. The rate of polymerdisintegration can vary from several weeks to periods of greater thanone year, depending on several factors including polymer molecularweight, ratio of lactide to glycolide monomers in the polymer chain, andstereoregularity of the monomer subunits (mixtures of L and Dstereoisomers disrupt the polymer crystallinity enhancing polymerbreakdown). Particularly useful results are obtained by blending PLGAhaving different molecular weights, and/or different ratios of lactideto glycolide. The molecular weight and monomer ratios can be optimizedto tailor the release kinetics over a defined period of time. The highermolecular weights, result in polymer matrices which retain theirstructural integrity for longer periods of time; while lower molecularweights, result in both faster release and shorter matrix lives.

In a preferred embodiment described herein, the microspheres containblends of at least two and more preferably three or more biodegradablepolymers, preferably hydrolytically unstable polymers, most preferablypoly(hydroxy acids) of different molecular weight and/or monomer ratio.In a preferred embodiment, three different molecular weight PLGAs areblended to form a composition that has linear release over a definedperiod of time, ranging from at least one day to about sixty days. In amore preferred embodiment to obtain release from about one to twenty-onedays, the PLGAs have molecular weights between 1000 and 20,000, morepreferably between 5,000 and 10,000, between 20,000 and 35,000, morepreferably between 25,000 and 30,000, and between 35,000 and 70,000,more preferably 5000 and 10,000. In the most preferred embodiment forrelease over a period of about one week, PLGAs having molecular weightsof about 6,000, 30,000, and 41,000 are combined.

PLA polymers are usually prepared from the cyclic esters of lacticacids. Both L(+) and D(−) forms of lactic acid can be used to preparethe PLA polymers, as well as the optically inactive DL-lactic acidmixture of D(−) and L(+) lactic acids. Methods of preparing polylactidesare well documented in the patent literature. The following U.S.patents, the teachings of which are hereby incorporated by reference,describe in detail suitable polylactides, their properties and theirpreparation: U.S. Pat. No. 1,995,970 to Dorough; U.S. Pat. No. 2,703,316to Schneider; U.S. Pat. No. 2,758,987 to Salzberg; U.S. Pat. No.2,951,828 to Zeile; U.S. Pat. No. 2,676,945 to Higgins; and U.S. Pat.Nos. 2,683,136; 3,531,561 to Trehu.

Since it is desirable to target delivery of GM-CSF to white cells,particularly in the case where the GM-CSF is being used as an adjuvant,alone or in combination with antigen, the polymer may be selected basedon properties other than just controlled release. For example, it isknown that certain polymers are inflammatory and therefore attractleukocytes, macrophages and other “white” cells. Examples of“chemoattractant” polymers include the polyhydroxy acids (PL, PG,PLGAs), polyanhydrides, poly(ortho esters), and the polyphosphazenes.

In the case where the microparticles are intended for transmucosal ororal delivery, it may be desirable to select polymers which arebioadhesive. Examples of bioadhesive polymers include hydrophilicpolymers, especially those containing carboxylic groups, such aspoly(acrylic acid). Rapidly bioerodible polymers such aspoly(lactide-co-glycolide), polyanhydrides, and polyorthoesters havingcarboxylic groups exposed on the external surface as their smoothsurface erodes, are particularly useful. Representative natural polymersare proteins, such as zein, albumin, and collagen, and polysaccharides,such as cellulose, dextrans, and alginic acid. Other representativesynthetic polymers include polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, and nitrocelluloses,polymers of acrylic and methacrylic esters, poly(lactide-co-glycolide),polyanhydrides, polyorthoesters blends and copolymers thereof.

Polymers for Formation of Hydrogels

Other polymeric materials that may be useful include hydrogels such asthe naturally occurring polysaccharides like alginate, as well assynthetic hydrogel materials such as some of the polyacrylic acids,polyphosphazenes, polyethylene glycol-PIGA copolymers and othersynthetic biodegradable polymers which absorb up to 90% of the finalweight of water.

The polymeric material which is mixed with GM-CSF for implantation intothe body should form a hydrogel. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate, polyphosphazenes, and polyacrylates,which are crosslinked ionically, or block copolymers such as Pluronics™or Tetronics™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively. Othermaterials include proteins such as fibrin, polymers such aspolyvinylpyrrolidone, hyaluronic acid and collagen. U.S. Pat. Nos.5,286,495 and 5,410,016 to Hubbell, et al., describe useful materialsfor forming biocompatible hydrogels.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and is sulfonated polymers, such assulfonated polystyrene. Copolymers having acidic side groups formed byreaction of acrylic or methacrylic acid and vinyl ether monomers orpolymers can also be used. Examples of acidic groups are carboxylic acidgroups, sulfonic acid groups, halogenated (preferably fluorinated)alcohol groups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Calcium alginate and certain other polymers can form ionic hydrogelswhich are malleable can be used to encapsulate GM-CSF. Alginate can beionically cross-linked with divalent cations, in water, at roomtemperature, to form a hydrogel matrix. The hydrogel is produced bycross-linking the anionic salt of alginic acid, a carbohydrate polymerisolated from seaweed, with divalent cations, whose strength increaseswith either increasing concentrations of calcium ions or alginate.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N⁺—C₆—^(+NR) ₃ can also be used. Aqueoussolutions of the salts of these cations are added to the polymers toform soft, highly swollen hydrogels and membranes. The higher theconcentration of cation, or the higher the valence, the greater thedegree of cross-linking of the polymer. Concentrations from as low as0.005 M have been demonstrated to cross-link the polymer. Higherconcentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of the polymers to form ahydrogel are divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan. Polyanions that can beused to form a semi-permeable membrane by reaction with basic surfacegroups on the polymer hydrogel include polymers and copolymers ofacrylic acid, methacrylic acid, and other derivatives of acrylic acid,polymers with pendant SO₃H groups such as sulfonated polystyrene, andpolystyrene with carboxylic acid groups.

These polymers are either commercially available or can be synthesizedusing methods known to those skilled in the art. See, for exampleConcise Encyclopedia of Polymer Science and Polymeric Amines andAmmonium Salts, E. Goethals, editor (Pergamen Press, Elmsford, N.Y.1980).

GM-CSF

GM-CSF, granulocyte macrophage colony stimulating factor, is ahematopoietic growth factor which promotes the proliferation anddifferentiation of hematopoietic progenitor cells. The cloned gene forGM-CSF has been expressed in bacteria, yeast and mammalian cells. Theendogenous protein is a monomeric glycoprotein with a molecular weightof about 22,000 daltons. The recombinant preparation expressed inbacterial cells is unglycosylated GM-CSF produced in a yeast expressionsystem is marketed as leukine® by Immunex Corporation, Seattle, Wash.Leukine™ is sold in lyophilized form. It is a glycoprotein of 127 aminoacids characterized by three primary molecular species having molecularmasses of 19,500, 16,800, and 15,500 daltons.

GM-CSF is described in U.S. Pat. No. 5,078,996 to Conlon, et al. Analogsof GM-CSF are described in U.S. Pat. Nos. 5,229,496, 5,393,870, and5,391,485 to Deeley, et al. In the preferred embodiment the GM-CSF isrecombinant protein having a molecular weight of between approximately14,000 and 20,000, made in yeast which hyperglycosylates the proteinpresumably limiting the amount of non-specific absorption observed withthe protein. GM-CSF fusion proteins can also be used. Examples withGM-CSF fusion proteins include fusion proteins with IL-3 and otherlymphokines or growth factors.

Preparation of Microparticles

Microspheres, or solid microparticles, can be prepared using any of anumber of techniques known to those skilled in the art. GM-CSF appearsto be unusually stable to processing, especially in the presence oforganic solvents, which facilitates microparticle formation containingGM-CSF having very high levels of bioactivity, typically greater than90% as compared to the GM-CSF prior to incorporation into themicroparticles. Examples of methods for preparation include solventevaporation, spray drying, solvent extraction and other methods known tothose skilled in the art. As discussed above, hydrogels are typicallyformed by ionic crosslinking, by addition of ions or polyions, orphotocrosslinking or other forms of chemical crosslinking.

Microsphere Preparation

Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release 5,13-22(1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988), theteachings of which are incorporated herein. The selection of the methoddepends on the polymer selection, the size, external morphology, andcrystallinity that is desired, as described, for example, by Mathiowitz,et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J.Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm.Sci. 73, 1721-1724 (1984), the teachings of which are incorporatedherein. Methods include solvent evaporation, phase separation, spraydrying, and hot melt encapsulation. U.S. Pat. Nos. 3,773,919; 3,737,337;and 3,523,906 are representative of methods for making microspheres.

A preferred method is described in U.S. Pat. No. 5,000,886 to Lawter, etal., the teachings of which are incorporated herein. The GM-CSF isdispersed in an aqueous solution which is then mixed with an organicsolution of the polymer. The dispersion is added to a non-solvent forthe polymer and the GM-CSF, then the microparticles hardened byextraction of the polymer solvent into a volatile silicone fluid.

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent. The GM-CSF, either in solubleform or dispersed as fine particles, is added to the polymer solution,and the mixture is suspended in an aqueous phase that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporates, leaving solidmicrospheres.

In general, the polymer can be dissolved in methylene chloride. Severaldifferent polymer concentrations can be used, for example, between 0.01and 0.50 g/ml. After loading the solution with GM-CSF, the solution issuspended in 200 ml of vigorously stirring distilled water containing 1%(w/v) poly(vinyl alcohol) (Sigma Chemical Co., St. Louis, Mo.). Afterfour hours of stirring, the organic solvent will have evaporated fromthe polymer, and the resulting microspheres will be washed with waterand dried overnight in a lyophilizer.

Microspheres with different sizes (1-1000 microns) and morphologies canbe obtained by this method which is useful for relatively stablepolymers such as polyesters and polystyrene. However, labile polymerssuch as polyanhydrides may degrade due to exposure to water. For thesepolymers, hot melt encapsulation and solvent removal may be preferred.

Solvent removal is particularly useful with polyanhydrides. In thismethod, the drug is dispersed or dissolved in a solution of a selectedpolymer in a volatile organic solvent like methylene chloride. Themixture is then suspended in oil, such as silicone oil, by stirring, toform an emulsion. Within 24 hours, the solvent diffuses into the oilphase and the emulsion droplets harden into solid polymer microspheres.Unlike solvent evaporation this method can be used to make microspheresfrom polymers with high melting points and a wide range of molecularweights. Microspheres having a diameter between one and 300 microns canbe obtained with this procedure. The external morphology of the spheresis highly dependent on the type of polymer used.

In spray drying, the polymer is dissolved in methylene chloride (0.04g/ml). A known amount of active drug is suspended (if insoluble) orco-dissolved (if soluble) in the polymer solution. The solution or thedispersion is then spray-dried. Microspheres ranging in diameter betweenone and ten microns can be obtained with a morphology which depends onthe selection of polymer.

Hydrogel microparticles made of gel-type polymers such as alginate orpolyphosphazenes or other dicarboxylic polymers can be prepared bydissolving the polymer in an aqueous solution, suspending the materialto be incorporated into the mixture, and extruding the polymer mixturethrough a microdroplet forming device, equipped with a nitrogen gas jet.The resulting microparticles fall into a slowly stirring, ionichardening bath, as described, for example, by Salib, et al.,Pharmazeutische Industrie 40-11A, 1230 (1978), the teachings of whichare incorporated herein. The advantage of this system is the ability tofurther modify the surface of the microparticles by coating them withpolycationic polymers such as polylysine, after fabrication, forexample, as described by Lim, et al., J. Pharm. Sci. 70, 351-354 (1981).As described by Lim, et al., in the case of alginate, a hydrogel can beformed by ionically crosslinking the alginate with calcium ions, thencrosslinking the outer surface of the microparticle with a polycationsuch as polylysine, after fabrication. The microsphere particle size arecontrolled using various size extruders, polymer flow rates and gas flowrates.

Chitosan microparticles can be prepared by dissolving the polymer inacidic solution and crosslinking with tripolyphosphate. For example,carboxymethylcellulose (CMC) microsphere are prepared by dissolving thepolymer in an acid solution and precipitating the microparticles withlead ions. Alginate/polyethylene imide (PEI) can be prepared to reducethe amount of carboxyl groups on the alginate microparticles.

Loading of GM-CSF

The range of loading of the GM-CSF to be delivered is typically betweenabout 0.001% and 10%, by weight. GM-CSF can be incorporated into apolymeric matrix at a ratio of between 0.001% by weight up to 10% byweight. In a preferred embodiment, GM-CSF is incorporated into PLGAblends to 2% by weight.

Loading is dependent on the disorder to be treated as well as the timeperiod over which the GM-CSF is to be released. Lower dosages arerequired for use as a vaccine adjuvant, in the range of 0.001 to 0.1%.Microparticles for treatment of a severe infection would typically bedelivered in microparticles with 2% by weight drug loading.

Additives to Microparticles Altering Release

Polymer hydrolysis is accelerated at acidic or basic pH's and thus theinclusion of acidic or basic excipients can be used to modulate thepolymer erosion rate. The excipients can be added as particulates, canbe mixed with the incorporated GM-CSF or can be dissolved within thepolymer.

Degradation enhancers are based on weight relative to the polymerweight. They can be added to the protein phase, added as a separatephase (i.e., as particulates) or can be codissolved in the polymer phasedepending on the compound. In all cases the amount should be between 0.1and thirty percent (w/w, polymer). Types of degradation enhancersinclude inorganic acids such as ammonium sulfate and ammonium chloride,organic acids such as citric acid, benzoic acids, heparin, and ascorbicacid, inorganic bases such as sodium carbonate, potassium carbonate,calcium carbonate, zinc carbonate, and zinc hydroxide, and organic basessuch as protamine sulfate, spermine, choline, ethanolamine,diethanolamine, and triethanolamine and surfactants such as Tween™ andPluronic™.

Pore forming agents are used to add microstructure to the matrices(i.e., water soluble compounds such as inorganic salts and sugars). Theyare added as particulates. The range should be between one and thirtypercent (w/w, polymer).

Excipients can be also added to the GM-CSF to maintain its potencydepending on the duration of release. Stabilizers include carbohydrates,amino acids, fatty acids, and surfactants and are known to those skilledin the art. In addition, excipients which modify the solubility ofGM-CSF such as salts, complexing agents (albumin, protamine) can be usedto control the release rate of the protein from the microparticles.

Stabilizers for the GM-CSF are based on the ratio by weight ofstabilizer to the GM F on a weight basis. Examples include carbohydratesuch as sucrose, lactose, manitol, dextran, and heparin, proteins suchas albumin and protaiine, amino acids such as arginine, glycine, andthreonine, surfactants such as Tween™ and Pluronic™, salts such ascalcium chloride and sodium phosphate, and lipids such as fatty acids,phospholipids, and bile salts.

The ratios are generally 1:10 to 4:1, carbohydrate to protein, aminoacids to protein, protein stabilizer to protein, and salts to protein;1:1000 to 1:20, surfactant to protein; and 1:20 to 4:1, lipids toprotein.

Clinical Indications to be Treated

Systemic Delivery for Proliferation of Cells

GM-CSF is approved for treatment of patients requiring increasedproliferation of white blood cells. Data indicates that GM-CSP is alsouseful as a vaccine adjuvant Morrissey, et al., J. Immunology 139,1113-1119 (1987). GM-CSF microparticles can also be used to treatpatients prone to infection such as those undergoing high risk bowelsurgery, trauma victims and individuals with HIV. The protocols andclinical efficacy of GM-CSF is well known to those skilled in the art.As described herein, the protocols are modified to reflect the changesin delivery rates and dosages resulting from the release profiles frommicroparticles or hydrogels, as appropriate.

In vitro data regarding release profiles for GM-CSF, as well asefficacy, appears to be predictive, although not identical, of in vivodata. As demonstrated by the following examples, Rhesus monkey data showmaximum increases in leucocyte numbers within four days followingadministration of GM-CSF, while in vitro results demonstrated that sixto seven days are required for complete release of the incorporatedGM-CSF. The advantage of using GM-CSF is that the protein is itselfextremely stable, with at least 60%, in many cases 90 to 100%, of thebioactivity being retained after incorporation into microparticles usingany one of several processes.

Local Administration as Adjuvant

Enhanced vaccine response can be obtained through the use of GM-CSFalone, but is more preferably obtained through a selection of thepolymer in combination with the controlled release of the GM-CSF. It isknown that certain polymers serve as chemoattractants for white cells.PLGA is mildly inflammatory, as are polyanhydrides and polyorthoesters.Through the selection of the chemoattractant polymer as the matrix forGM-CSF, in a form yielding controlled release over a period ofapproximately one week, maximum vaccine enhancement can be obtained. Inthis embodiment, release can be from polymeric matrices in a variety offorms not just microparticles or hydrogels. The GM-CSF and polymer mayeven act synergistically to enhance the adjuvant effect of the GM-CSF,as well as targeting of the GM-CSF to the white cells.

The GM-CSF can also be injected with a tumor antigen or tumor cells thatexpress antigens or their surfaces for use as a tumor vaccine.

Topical or Transmucosal Administration

The hydrogel formulations are particularly useful for topicalapplications. For example, GM-CSF has been shown by Braunstein et al.,J. Invest. Dermatol 103, 601-604 (1994) to stimulate keratinocyteproliferation in human skin and could thus be utilized as a topicalwound healing agent. Mucosal delivery of GM-CSF microparticles couldalso be efficacious in the stimulation of mucosal immunity since theprotein has been shown to play a role in antibody production (Grabstein,et al., J. Mol. Cell. Immunol. 2, 199-207 (1986)).

Administration of the GM-CSF Microparticles

In the preferred embodiment for stimulation of proliferation ofhematopoietic progenitor cells, GM-CSF is administered incorporated inmicroparticles which degrade over a period of 1 of 2 months. Themicroparticles preferably range in size from 10 to 60 microns, with anaverage of 35 microns in diameter, and are injected simultaneously withthe aid of a suspension media. In one embodiment, the suspension mediaconsists of 3% methyl cellulose, 4% mannitol, and 0.1% Tween™ 80, usinga 22 gauge needle. In another embodiment the 3% methylcellulose isreplaced by 1% carboxy methylcellulose. One ml of viscous suspensionmedia is required to suspend 100 milligrams of microparticles whichcontain enough GM-CSF to deliver 125 micrograms/m²/day over a period of7 days. Larger doses may be achieved by injecting more microparticles.For example, a 250 microgram/m²/day dose would require two 1 mlinjections, each containing 100 mg of microparticles.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1

Preparation of Microspheres Using Phase Separation Process.

A. tot #14223-133. Sample “A”

The encapsulating polymer was a poly(glycolide-d,l lactide) having aninherent viscosity of 0.43 ding (as determined in a 0.5% w/vhexafluoroisopropanol solution at 30° C.), and a glycolide to lactideratio of 45:55. It was prepared with glycolic acid as the initiator andstannous chloride dihydrate as the catalyst. The distribution of lactoyland glycoyl groups within the copolymer was shown to be random by C13NMR and solubility measurements. The residual lactide content wasreduced by vacuum stripping. The encapsulating polymer solution wasprepared by adding 100 g of the polymer to 900 g of methylene chloride,and stirring the mixture until the polymer dissolved.

0.978 ml of a GM-CSF solution (at 63.3 mg/ml in 100 mM tris buffer) wasadded to a 20 g portion of the encapsulating polymer solution. Themixture was stirred with a homogenizer using a 10-mm head at 10,000 RPMfor 2 minutes to create a water-in-oil (W/O) emulsion.

18.0 g of Dow Corning® 360 Fluid (polydimethylsiloxane). was added tothe W/O emulsion, and the mixture was homogenized at 10,000 RPM for 2minutes. The mixture was then added to 2.4 kg of Dow Corning, 244 Fluid(octamethylcyclotetrasiloxane) under stirring at 750 RPM to harden themicrospheres. Stirring was continued for 90 minutes. Microspheres werecollected with a strainer fitted with a 1 jam stainless steel screen,then dried under vacuum.

Particle size distribution was analyzed with a Malvern 2600 ParticleSizer. Approximately 50 mg of microspheres were suspended in about 10 mlof Dow Corning® 244 Fluid, and was sonicated for 2 minutes to fullydisperse the microspheres. A few drops of this suspension were thenadded to the optical cell which contained Dow Coming® 244 Fluid. Theparticle size distribution was then measured. The sample had a volumemedian diameter of 66.7 μm, 10% of the microspheres were under 24.1 μm,90% of the microspheres were under 118.6 μm.

B. Lot #14223-134. Sample “B”

The encapsulating polymer and its solution in methylene chloride werethe same as described in “A”.

0.320 ml of a GM-CSF solution (at 63.3 mg/mn in 100 mM tris buffer) wasadded to a 20 g portion of the encapsulating polymer solution. Themixture was stirred with a homogenizer using a 10-mm head at 10,000 RPMfor 2 minutes to create a water-in-oil (W/O) emulsion.

18.0 g of Dow Corning® 360 Fluid (polydimethylsiloxane) were added tothe W/O emulsion, and the mixture was homogenized at 10,000 RPM for 2minutes. The mixture was then added to 2.4 kg of Dow Corning® 244 Fluid(octamethylcyclotetrasiloxane) under stirring for 90 minutes at 750 RPMto harden the microspheres. Microspheres were collected, dried andparticle size distribution analyzed as described in “A”. The sample hada volume median diameter of 43.8 μm, 10% of the microspheres were under7.0 μm, 90% of the microspheres were under 77.9 μm.

As shown in FIG. 1A, samples “A” and “B” demonstrate that PLGAmicrospheres can be fabricated to release GM-CSF in a triphasic manner.In the first phase, the protein is released continuously overapproximately 5 days. This phase is followed by a period of minimalGM-CSF release until day 35. At this time another pulse of GM SF isreleased from the system. The duration of each phase can be controlledby the type of polymers used to prepare the microspheres.

C. Lot #9663-96A, Sample “B4”

The encapsulating polymer was a 60:20:20 mixture of 1) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of47:53 and an inherent viscosity of 0.72 dL/g as determined in a 0.5% w/vhexafluoroiisopropanol solution at 30° C. (polymer 1), 2) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ration of50:50 and an inherent viscosity of 0. 33 dL/g as determined in a 0.1%w/v chloroform solution at 25° C. (polymer II), and 3) a poly(d,llactide) with an average molecular weight of 1938 as determined by endgroup titration (polymer Ill). Polymer II and polymer III werereprecipitated before use. The encapsulating polymer solution wasprepared by adding 1.20 g of polymer I, 0.40 g of Polymer II, and 0.40 gof polymer III to 18.00 g of methylene chloride and stirring the mixtureuntil the polymers dissolved.

0.481 mld of a GM-CSF solution at 84.8 mg/ml in 100 mM tris buffer) wasadded to the encapsulating polymer solution, homogenized with a 20-mmhead at 10,000 RPM for 60 seconds to created a water-in-oil (W/O)emulsion.

The beaker containing the W/O emulsion was placed under a mixer equippedwith a 3-blade Teflon stirrer and stirred at 1000 RPM. 20 ml of DowCorning® 360 Fluid was added to the W/O emulsion while it was beingstirred at 1000 RPM over a 1-minute period of time using a syringe pump.

The mixture was then added to 2.0 kg of Dow Corning® 244 Fluid understirring for 90 minutes at 400 RPM to harden the microspheres.

Microspheres were collected, dried and Particle size distribution wasanalyzed as described above. The sample had a volume median diameter of31.8 μm, 1017, were under 14.1 μm and 90% of the microspheres were under52.2 μm.

D. Lot #9663-135C. Sample “04.”

The encapsulating polymer was a 60:20:20 mixture of: 1) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of47:53 and an inherent viscosity of 0.72 dL/g as determined in a 0.5% w/vhexafluoroisopropanol solution at 30° C. (polymer I), 2) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of50:50 and an inherent viscosity of 0.33 dL/g as determined in a 0.1% w/vchloroform solution at 25° C. (polymer II), and 3) a poly(d,l lactide)with an average molecular weight of 1938 as determined by end grouptitration (polymer III). Polymer II and polymer III were reprecipitatedbefore use. The encapsulating polymer solution was prepared by adding1.20 g of polymer I, 0.40 g of polymer II, and 0.40 g of polymer III to18.00 g of methylene chloride and stirring the mixture until thepolymers dissolved.

0.462 ml of a GM-CSF solution (at 88.4 mg/ml in 100 mM tris buffer) wasadded to the encapsulating polymer solution, homogenized with a 20-mmhead at 10,000 RPM for 60 seconds to create a water-in-oil (W/O)emulsion. The W/O emulsion was stirred at 1000 RPM. 20 ml of DowCorning® 360 Fluid was added to the W/O emulsion while it was beingstirred over a 1-minute period of time using a syringe pump. The mixturewas then added to 2.0 kg of Dow Corning® 244 Fluid under stirring at 400RPM for 90 minutes to harden the microspheres.

Microspheres were collected, dried and particle size distribution wasanalyzed as described above. The sample had a volume median diameter of39.5 μm, 10% of the microspheres were under 14.9 μm, and 90% of themicrospheres were under 65.1 μm.

E. Lot #9490-168. Sample “V4.” Aseptic Process

Microspheres were prepared aseptically as follows. Glassware, mixershafts and heads, and stainless steel vessels were autoclaved prior touse.

The encapsulating polymer was a 60:20:20 mixture of 1)poly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of47:53 and an inherent viscosity of 0.72 dL/g as determined in a 0.5% w/vhexafluoroisopropanol solution at 30° C. (polymer I), 2) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of50:50 and an inherent viscosity of 0.33 dL/g as determined in a 0.1% w/vchloroform solution at 25° C. (polymer II), and 3) a poly(d,l lactide)with an average molecular weight of 1938 as determined by end grouptitration (polymer III). Polymer II and polymer III were reprecipitatedbefore use. The encapsulating polymer solution was prepared by adding3.00 g of polymer I, 1.00 g of polymer II, and 1.00 g of polymer III to45.00 g of methylene chloride and stirring the mixture until thepolymers dissolved. 20.00 g of this solution was filtered through aglass fiber prefilter and a 0.45 μm Teflon filter formicroencapsulation.

Approximately 100 g of Dow Corming® 360 Fluid was heated at 160° C. for1 hour in a glass beaker covered with aluminum foil, then cooled to roomtemperature.

2.0 kg of Dow Corning® 244 Fluid was filtered through a “Millipak™ 20”0.22 μm filter into the hardening vessel.

0.485 ml of a GM-CSF solution (at 87.3 mg/ml in 100 mM tris buffer,filtered through a 0.2 μm filter) was added to the 20 grams of filteredencapsulating polymer solution, stirred with a homogenizer using a 20-mmhead at 10,000 RPM for 60 seconds to create a water-in-oil (W/O)emulsion.

The beaker containing the W/O emulsion was placed under a mixer equippedwith a 3-blade Teflon stirrer and stirred at 1000 RPM. 20 ml of theheat-treated Dow Corning® 360 Fluid was added to the W/O emulsion whileit was being stirred at 1000 RPM over a 1 minute period of time using asyringe pump.

The mixture was then added to the 2.0 kg of filtered Dow Corning® 244Fluid under stirring at 400 RPM to harden the microspheres. Stirring wascontinued for 90 minutes.

Microspheres were collected, dried and the particle size distributionwas determined with a Malvern 2600 Particle Sizer. The particle sizedistribution was then measured. The sample had a volume median diameterof 32.5 μm, 10% of the microspheres were under 14.7 μm, and 90% of themicrospheres were under 54.8 μm.

EXAMPLE 2

Analysis of GM-CSF Incorporated into Microspheres Using Reversed PhaseHigh Performance Liquid Chromatography.

GM-CSF was first extracted from microspheres using acetic acid,methylene chloride and phosphate buffered saline. A control consistingof blank microspheres with freshly added stock GM-CSF was concurrentlyextracted. The extracts were then evaluated by RP-HPLC for glycoformdistribution.

Recombinant human (rhu) GM-CSF glycosylated variants are measured usinghigh performance liquid chromatography (HPLC). The column is a C18 10micron, 300 angstrom column (4.6 mm×25 cm) from Vydac. The glycoforms ofGM-CSF can be resolved in this method using a mobile phase gradient of25 to 65% 0.1% trifluoroacetic acid (TFA)/acetonitrile in 0.1% TFA/waterwith constant 200 mM NaCl.

Results show that the GM-CSF glycoform distribution of the control isnot altered due to the extraction protocol and the RP-HPLC profile ofGM-CSF incorporated into microspheres remains unchanged.

EXAMPLE 3

GM-CSF Release Kinetics from Microsphere Preparations.

Release kinetics of GM-CSF microspheres were analyzed using thefollowing methods. Microsphere lots were stored in their originalcontainers at 2-8° C. The in vitro release studies were initiated withina few days of preparation. Studies were set up in duplicate forscreening and in triplicate for increased confidence in accuracy andprecision. The release buffer was phosphate buffered saline (PBS, 12 mMsodium phosphate, 3.7 mM potassium phosphate, 150 mM sodium chloride, pH7.0, 0.22 μm filtered) containing 0.02% sodium azide as preservative,although it is recommended to leave out the azide and handle the releasestudy as aseptically as possible when bioactivity analysis of thereleased GM-CSF is planned. Collection time intervals in a study mayinclude times at 2, 6, 24, 48, 72 hrs, 5, 7, 10, and 14 days.

To set up an in vitro release assay, approximately 50 to 75 mg ofmicrospheres were placed in the 9 mm ID×13 mm OD×5 mm teflon spacerwhich is within the teflon jacket of the device. The spacer was anchoredat both ends with 13 mm diameter, 10 μm mesh stainless steel screenswhich allow circulation of release buffer through the microspheres.

The loaded device was placed into a wide, 30 ml polypropylene test tubewith 6 ml release buffer. The open tubes containing the loaded deviceand release buffer were covered with loose caps or Kimwipes® securedwith rubber bands and placed in a vacuum desiccator. Exposure to vacuumfor approximately 2 hours helps remove any trapped air bubbles andpromotes initial wetting of the microspheres. The tubes were then cappedand incubated at 37° C. on an orbital shaker set at 100 rpm.

At appropriate time intervals the release buffer was retrieved bydecanting into preweighed, labelled 15 ml polypropylene test tubes. Thevolume obtained was determined by weight (ml=g, approximately). Thedevice, with an addition of fresh release buffer (6 ml), was thenreturned to the orbital shaker at 37° C.

The microsphere release samples were evaluated by the Bio-Rad TotalProtein Assay to quantify the release of GM-CSF. A total protein assaycan be used since there is no other protein present.

The results of the release kinetics analysis are shown in FIGS. 1B and3A. Samples “B4”, “04” and “V4”, prepared in Example 1, show that PLGAmicrospheres can be fabricated to continuously release GM-CSF over aperiod of approximately 6 days. The release is linear over this time andapproaches zero order. Furthermore, the GM-CSF is released withessentially complete retention of bioactivity as shown in FIG. 3B (giventhe standard deviation of the bioassay, the released GM-CSF can beconsidered completely active.) These examples also show that themicrosphere fabrication process is highly reproducible as evidenced bythe very similar GM-CSF release profiles generated from the threepreparations.

EXAMPLE 4

In Vivo Release Profile of huGM-CSF in a Murine Model

A. Lot #9402-94, Sample “O”

Microspheres were prepared aseptically as follows:

Glassware, mixer shafts and heads, and stainless steel vessels wereautoclaved prior to use. 4.0 kg of Dow Corning® 244 Fluid(octamethylcyclotetrasiloxane) was filtered through a “Millipak™ 40”0.22 μm filter into the hardening vessel. Approximately 100 g of DowComning® 360 Fluid (polydimethylsiloxane, 350 centistrokes) was heatedat 160° C. for 80 minutes in a glass beaker covered with aluminum foil,then cooled to room temperature.

A 40-gram portion of a 10% poly(glycolide-co-d,l lactide) solution inmethylene chloride was filtered through a polyvinylidene fluoride 0.22μm filter for microencapsulation. The polymer had an inherent viscosityof 0.43 dL/g as determined in a 0.5% w/v hexafluoroisopropanol solutionat 30° C., and a glycolide to lactide ratio of 45:55. It was preparedwith glycolic acid as the initiator and stannous chloride dihydrate asthe catalyst. The distribution of lactoyl and glycoyl groups within thecopolymer was shown to be random by C13 NMR and solubility measurements.The residual lactide content was reduced by vacuum stripping.

0.664 ml of a GM-CSF solution (about 63.1 mg/ml in 100 mM tris buffer,filtered through a 0.2 μm filter) was added to the 40 grams of filteredpolymer solution, s with a homogenizer using a 20 mm head at 10,000 RPMfor 60 seconds to create a water-in-oil (W/O) emulsion. 36 ml of theheat-treated Dow Corning® 360 Fluid was added to the W/O emulsion, andthe mixture was homogenized at 5000 RPM for 90 seconds. The mixture wasthen added to the 4.0 kg of filtered Dow Corning® 244 Fluid understirring at 500 RPM to harden the microspheres. Stirring was continuedfor 1 hour. Microspheres were collected, dried and particle sizedistribution analyzed as described above. The sample had a volume mediandiameter of 56.0 μm, 10% of the microspheres were under 26.3 μm, 90% ofthe microspheres were under 91.6 μm.

The microspheres were first analyzed for in vitro releasecharacteristics to ensure continuous release of huGM-CSF over a periodof greater than 7 days (FIG. 2A). The microspheres were then weighed andloaded into 3 cc syringes with 50 mg of microspheres/syringe. Theinjection vehicle for the microspheres was an aqueous solution of lowviscosity grade methyl cellulose, containing 3% (w/w) methyl cellulose,0.1% Tween 80, and 4% mannitol (final osmolality=292 mOsmfkg). Vialswere loaded with 1 g each of sterile filtered injection vehiclesolution. For injection, an 18-gauge needle was attached to an empty 3cc syringe and used to withdraw 0.5-0.6 ml of injection vehicle. Theneedle was then removed from the syringe and the syringe containing thevehicle was attached to a syringe containing microspheres through a“syringe connector”. Mixing was achieved by pushing the syringesback-and-forth 25 times in each direction. The empty syringe and thesyringe connector were then removed. A 22 gauge needle was then attachedto the syringe with suspended microspheres ready for injection.

Release Studies in Mice

Release studies were conducted on mice as follows. Male B6 mice (6 weeksold) were obtained from Jackson Laboratories (Bar Harbor, Me.) and werehoused in the Immunex animal laboratory facility for an additional 10weeks prior to initiating the study. Seventeen groups of mice were usedin the study, with three mice used per group. For the test group 50 mgof microspheres containing 500 μg of huGM-CSF (1% by wt) were injectedsubcutaneously in 0.5 ml of the methyl cellulose injection vehicle.Groups of mice receiving these injections were sacrificed at intervalsof 1, 2, 6, 24 hr, 3, 5, 7, and 9 days. As a negative control, one groupof mice was sacrificed without receiving huGM-CSF in any form. As afinal control a bolus of huGM-CSF was injected subcutaneously at a doseof either 500 or 50 μg of huGM-CSF. The 500 μg dose represented theentire amount of huGM-CSF contained in a 50 mg injection ofmicrospheres, and the 50 μg dose represented an approximation of theamount of huGM-CSF released by 50 mg of microspheres over a period of 1day in vitro. Groups of mice receiving bolus injections were sacrificedat intervals of 1, 2, 6, and 24 hr post-injection.

Following sacrifice of the mice, blood samples were obtained and allowedto clot at 4° C. The sera was then harvested and the clot was discarded.Remaining cellular debris was removed by centrifugation, and the serumsamples were then frozen at −70° C. until further analysis.

TABLE 1 In Vivo Microsphere Release Study Outline Post-Injection GroupDescription Sacrifice Time  1 no injection, negative control 0 hr  2 500μg huGM-CSF bolus injection 1 hr  3 50 μg huGM-CSF bolus injection 1 hr 4 50 mg microspheres injected 1 hr  5 500 μg huGM-CSF bolus injection 2hr  6 50 μg huGM-CSF bolus injection 2 hr  7 50 mg microspheres injected2 hr  8 500 μg huGM-CSF bolus injection 6 hr  9 50 μg huGM-CSF bolusinjection 6 hr 10 50 mg microspheres injected 6 hr 11 500 μg huGM-CSFbolus injection 24 hr 12 50 μg huGM-CSF bolus injection 24 hr 13 50 mgmicrospheres injection 24 hr 14 50 mg microspheres injected 72 hr (3day) 15 50 mg microspheres injected 120 hr (5 day) 16 50 mg microspheresinjected 168 hr (7 day) 17 50 mg microspheres injected 216 hr (9 day)

Serum samples were thawed and analyzed by ELISA for determination ofhuGM-CSF concentrations. The GM-CSF enzyme linked immunoassay (EIA) isan assay designed to quantitate levels of recombinant human (rhu) GM-CSFin an unknown sample. An anti-GM-CSF murine monoclonal antibody isadsorbed onto a 96-well polystyrene plate overnight. After washing, astandard curve and samples are added to the plate and incubated. Theplate is washed to remove any excess unabsorbed rhu-GM-CSF. A polyclonalantibody to rhu-GM-CSF is then added to each well and incubated. Theplate is washed to remove any unbound polyclonal antibody and a solutioncontaining donkey anti-sheep IgG antibody conjugated to horseradishperoxidase (HRP) enzyme is added to each well. Following incubation theplate is washed to remove any excess HRP-linked antibody which did notbind to the sheep antibodies present. A developing solution containingthe chromogenic substrate for the HRP conjugate is added to the plate.Color development is directly proportional to the amount ofHRP-conjugate present. The optical density readings at the correctwavelength give numerical values for each well. These wells can becompared with the standard curve values, permitting quantitation of thelevels of rhu-GM-CSF. A monoclonal anti GM-CSF antibody can be obtainedfrom Immunex. A donkey anti-sheep IgG antibody ImP conjugate is obtainedfrom Jackson Immunoresearch Laboratories.

Serum samples were also analyzed for bioactivity by the cellproliferation assay TF-1. A TF1 bioassay is used to detect the presenceand amount of human GM-CSF. The TF1 bioassay utilizes a humanerythroleukemia cell line, TF1, to detect the presence of huGM-CSF, huIL-3, or rhu PIXY321 in test samples. These cells are dependent uponhuGM-CSF for growth and are maintained in medium supplemented withhuGM-CSF. The addition of huGM-CSF, hu IL-3, or rhu PIXY321 to thesecells stimulates a dose-dependent proliferation, allowing forquantitation of huGM-CSF, hu IL-3, or rhu PIXY321 in test samples ascompared to a standard of known huGM-CSF, hu IL-3, or rhu PIXY321concentration.

The amount of proliferation is measured by “pulsing” each microwell withtritiated thymidine (³H-TdR) for 4 hours at 37° C. Proliferating TF-1cells will incorporate (³H-TdR) which is added to the medium into theirDNA as they divide. The cells from each well are then harvested onto aglass fiber filter paper which traps the labeled GM-CSF. The amount of³H-TdR trapped on each filter paper is then counted on a beta counter.The number of counts per minute (cpm) for each well is directlyproportional to the amount of proliferation by the activated TF-1 cellsin response to huGM-CSF, hu IL-3, or ru PIXY321. The resulting countsper minute are directly proportional to the amount of GM-CSF that wasstimulating the cell colony.

To determine bioactivity of GM-CSF in the release samples all samplesare diluted to 0.2 ng GM-CSF/ml and submitted for analysis. Theresulting activities expressed as units/ml are compared to the activityof an untreated stock sample of GM-CSF analyzed simultaneously. Intheory, all samples should have approximately 100% activity relative tothe stock sample. Between assays control values can range around 20 to25%, a precision level not unusual with assays based on cell growth.

The percentage of specific activity retained at each time point wasdetermined by dividing the specific activity measured at each time pointby the specific activity of stock huGM-CSF of the same lot which had notbeen incorporated into microspheres.

Results of the release study are shown in FIG. 2A, which is a graph ofthe in vitro release kinetics showing release over a period of about tendays. FIG. 2B is a graph of the circulating mouse serum huGM-CSF levels(determined by ELISA) as a function of time. Both the 500 and 50 μgbolus injections were rapidly cleared from mouse serum. Due to the rapiddecline of detectable huGM-CSF in mouse serum only a rough estimate ofthe β elimination half-life could be made (t_(1/2)β=1.57 hr); however,this estimate agrees closely with previously reported half-lives forhuGM-CSF circulating in a mouse model. Levels of serum huGM-CSF in themice which received microspheres dropped rapidly over the first 6 hourspost-injection (from 218 to 5 to 35 ng/ml), and then remained relativelyconstant over the remaining 9 days of the study. Presumably, given thein vitro release profile for this lot of microspheres (approximately 30%release after 9 days) huGM-CSF would have released from the microspheresbeyond the 9 day period where the in vivo study was terminated.

As shown in FIG. 2C, the in vivo release data was compared to the invitro release data as follows: (1) the in vitro release data was firstmathematically modeled to fit a power series; (2) a theoretical in vivoserum huGM-CSF concentration profile was then calculated by taking amass balance in a single compartment model (i.e. the huGM-CSFconcentration in the mouse at any time equals the concentration ofhuGM-CSF already in the mouse at a previous time point plus the amountof huGM-CSF released from the microspheres over that time period minusthe amount of huGM-CSF cleared by normal physiological clearancemechanisms over that same time period). The resulting comparison ofserum concentration based on in vitro release and actual in vivo serumhuGM-CSF levels is shown in FIG. 2C. As demonstrated in this figure theactual in vivo serum huGM-CSF levels were lower than those predicted bythe in vitro release, however, the profiles were similar in shape andremarkably close in values at later time points.

The bioactivity of huGM-CSF released from microspheres in vivo wasestimated by TF-1 bioassay. The percent of specific bioactivity variedfrom a high of 67% at 1 hour and gradually declined to a low of 33%after 9 days.

EXAMPLE 5

Release of Human GM-CSF from Microspheres in a Primate Model.

Microspheres containing huGM-CSF were prepared for in vivo injection ina rhesus monkey model (Lot #9490-168, sample “V4”). Microspheres werefirst characterized in vitro for protein loading (1.48% wt/wt by aminoacid analysis), release kinetics (see FIG. 3A) and bioactivity ofreleased material by TF-1 bioassay (see FIGS. 1B and 3B). Based on thein vitro release profile, microspheres were weighed out such thatprimates would receive approximately 25 μg/kg/day for 7 days. Syringeswere loaded with microspheres which included an extra 5% for the hold-upvolume encountered on injection (50 μl holdup volume for 1 cc tuberculinsyringe). Three primates received injections with microspherescontaining GM-CSF. One primate received placebo microspheres which didnot contain microspheres. The quantity of microspheres injected intoeach of the primates was 39.4 mg, 35.5 mg, 42.1 mg, and 36.8 mg, for 3.2kg, 2.9 kg, 3.4 kg, and 3.0 kg animals, respectively

The injection vehicle for the microspheres was an aqueous solution oflow viscosity grade methyl cellulose, containing 3% (wt/wt) methylcellulose, 0.1% Tween 80, and 4% mannitol.

Serum samples were collected and analyzed for white blood cell count(WBC), absolute neutrophil count (ANC), and platelet count on days 3 and1 prior to injection, on the day of injection, and daily following theinjection for 10 days. Daily blood cells counts are shown in FIG. 3C.

The WBCs and ANCs were clearly elevated on days 1 through 4 in each ofthe animals receiving GM-CSF containing microspheres. No changes inblood cell counts were measured for the primate receiving the placeboinjection.

This example shows that recombinant human GM-SCF released from PLGAmicrospheres in vivo, is capable of eliciting a biological response in anon-human primate model.

Highly localized inflammatory response seen in the monkeys wascharacterized by a significant localized swelling (1-2 cm diameter lump)at the site of injection as a result of recruitment of neutrophils,macrophages, dendritic cells and monocytes.

EXAMPLE 6

Release of GM-CSF From a PLGA Gel.

A 20% solution of PLGA (50:50 lactide glycolide ratio, 0.38 dL/gintrinsic viscosity (I.V.)) was prepared by heating 2 g of PLGA in 8 gof glycerol triacetate (triacetin) at 70° C. for 30 minutes. LyophilizedGM-CSF was added to the PLGA solution at 10 mg/ml and sonicated tocomplete mixing. Screw top vials (5 ml) were filled with 3 ml PBS andapproximately 250 μg of the PLGA/GM-CSF solution (containingapproximately 2.5 mg of GM-CSF) was added to each vial by pipetting. Thevials were shaken gently at 37° C. for 6 days. The injected solutionformed a gel on injection into the PBS. At intervals of 4 hr, 8 hr, 1day, 3 days, and 6 days, the solutions were removed from the vials andanalyzed for GM-CSF content by a BioRad total protein assay.

The protein release kinetics are shown in FIG. 4 and show first orderkinetics over a period of about six days.

EXAMPLE 7

Preparation of Microspheres Using a W/O/W-Methanol Extraction Process.(Lot #14254-138)

The encapsulating polymer was a 70:20:10 mixture of 1) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of50:50 and an inherent viscosity of 0.33 dL/g as determined in a 0.1% w/vchloroform solution at 25° C. (polymer I), 2) a poly(L-lactide) with anaverage molecular weight of 1786 as determined by end group titration(polymer II), and 3) a poly(d,l lactide) with an average molecularweight of 1938 as determined by end group titration (polymer II). Thepolymers were reprecipitated before use. The encapsulating polymersolution was prepared by adding 1.40 g of polymer I, 0.40 g of polymerII, and 0.20 g of polymer III to 8.00 g of methylene chloride andstirring the mixture until the polymers dissolved.

A 5% aqueous solution of polyvinyl alcohol (PVA) was prepared by adding20.00 g of low molecular weight (M.W.=31,000-50,000, 87-89% hydrolyzed)PVA to 380 g of deionized water, and stirring with heating (toapproximately 70° C.) until the PVA is dissolved. The solution wasfiltered through a 0.2 μm filter after cooling to room temperature.

0.389 ml of a GM-CSF solution (about 87.3 mg/ml in 100 mM tris buffer)was added to 8.50 g of the encapsulating polymer solution in a 30-mlglass beaker, and was homogenized with a 20mm head at 6000 RPM for 60seconds to create a water-in-oil (W/O) emulsion.

The above emulsion was added to 400 g of the 5% PVA solution in astainless steel vessel while it was being stirred with a homogenizer at6000 RPM using a 20-mm head to create a water-in-oil-in-water (W/O/W)emulsion. Total elapsed time was 1 minute.

The vessel containing the W/O/W emulsion was placed under a mixerequipped with a “high shear disperser” and stirred at 400 RPM. 400 g ofmethanol was added to the W/O/W emulsion over a 45-minute period toextract the methylene chloride from the microspheres. Stirring wascontinued for another 45 minutes after the addition of methanol.

Microspheres were collected, dried and particle size distribution wasdetermined as described above.

The sample had a volume median diameter of 24.1 μm, 10% of themicrospheres were under 10.8 μm, and 90% of the microspheres were under42.3 μm.

EXAMPLE 8

Microsphere Preparation using a W/O/W Methanol Extraction Process. (Lot#14254-160)

The encapsulating polymer was a 80:10:10 mixture of 1) apoly(glycolide-co-d,l lactide) having a glycolide to lactide ratio of50:50 and an inherent viscosity of 0.33 dL/g as determined in a 0.1% w/vchloroform solution at 25° C. (polymer 1), 2) a poly(L-lactide) with anaverage molecular weight of 1786 as determined by end group titration(polymer II), and 3) a poly(d,l lactide) with an average molecularweight of 1938 as determined by end group titration (polymer III). Thepolymers were reprecipitated before use. The encapsulating polymersolution was prepared by adding 1.60 g of polymer I, 0.20 g of polymerII, and 0.20 g of polymer III to 8.00 g of methylene chloride andstirring the mixture until the polymers dissolved.

A 5% aqueous solution of polyvinyl alcohol (PVA) was prepared by adding20.00 g of low molecular weight (M.W.=31,000-50,000, 87-89% hydrolyzed)PVA to 380 g of deionized water, and stirring with heating (toapproximately 70° C.) until the PVA is dissolved. The solution wasfiltered through a 0.2 μm filter after cooling to room temperature.

0.389 ml of a GM-CSF solution (about 87.3 mg/ml in 100 mM Tris buffer)were added to 8.50 g of the encapsulating polymer solution in a 30-mlglass beaker, and was homogenized with a 20-mm head at 6000 RPM for 60seconds to create a water-in-oil (W/O) emulsion.

The above emulsion was added to 400 g of the 5% PVA solution in astainless steel vessel while it was being stirred with a homogenizer at6000 RPM using a 20-mm head to create a water-in-oil-in-water (W/O/W)emulsion. Total elapsed time was 1 minute.

The vessel containing the W/O/W emulsion was placed under a mixerequipped with a “high shear disperser” and stirred at 400 RPM. 400 g ofmethanol was then pumped into the W/O/W emulsion at a constant rate overa 5-minute period to extract the methylene chloride from themicrospheres. Stirring was continued for another 85 minutes after theaddition of methanol.

Microspheres were collected, dried, and particle size distribution wasdetermined as described above.

The sample had a volume median diameter of 26.1 μm, 10% of themicrospheres were under 11.6 μm, 90% of the microspheres were under 46.7μm.

EXAMPLE 9

Preparation of microspheres Lot #14259-100 (Hydrogel)” by a W/O/Wmethanol extraction process.

The encapsulating polymer was a 67:23:10 block tripolymer ofcaprolactone, trimethylene carbonate, and polyethylene oxide 8000. 3.27g of the polymer was mixed with 18.53 g of methylene chloride andstirred until the polymer dissolved.

A 1% aqueous solution of polyvinyl alcohol (PVA) was prepared by adding11.0 g of low molecular weight (M.W.=31,000-50,000, 87-89% hydrolyzed)PVA to 1089.00 g of deionized water, and stirring with heating (toapproximately 70° C.) until the PVA dissolved. The solution was filteredthrough a 0.2 μm filter after cooling to room temperature.

0.174 ml of a GM-CSF solution (at 87.1 mg/ml in 100 mM Tris buffer) wasadded to a 10.00 g portion of the encapsulating polymer solution in a30-ml glass beaker, and was homogenized with a 20-mm head at 6000 RPMfor 60 seconds to create a water-in-oil (W/O) emulsion.

The above emulsion was added to a 500 g portion of the 1% PVA solutionin a stainless steel vessel while it was being stirred with ahomogenizer at 6000 RPM using a 20-mm head to create awater-in-oil-in-water (W/O/W) emulsion. Total elapsed time was 1 minute.

The vessel containing the W/O/W emulsion was placed under a mixerequipped with a “high shear disperser” and stirred at 400 RPM. 500 g ofmethanol was pumped into the W/O/W emulsion over a 5-minute period toextract the methylene chloride from the microspheres. Stirring wascontinued for another 55 minutes after the addition of methanol.

Microspheres were collected, dried and particle size distribution wasdetermined with a Malvern 2600 Particle Sizer. Approximately 50 mg ofmicrospheres was suspended in about 10 ml of methanol, and was sonicatedfor 2 minutes to fully disperse the microspheres. A few drops of thissuspension were then added to the optical cell which contained methanol.The particle size distribution was then measured. The sample had avolume median diameter of 68.3 um, 10% of the microspheres were under14.3 im and 90% of the microspheres were under 177.3 im.

EXAMPLE 10

Extraction of GM-CSF from Microspheres for In vitro Determination ofBioactivity.

This method of extracting the protein from the microspheres is bothquantitative and nondestructive and therefore can be used to determinethe integrity of the incorporated protein. Approximately 20 mgmicrospheres (Lots L3, M3, N3, K3, J3, and E3) prepared by the phaseseparation method described in Example 1 were weighed into a 2 mlEppendorf tube. 500 μl of glacial acetic acid was then added and thetubes were capped and periodically vortexed to completely dissolve themicrospheres. 500 μl methylene chloride was then added and the tubeswere capped and vortexed periodically for about 5 minutes. Finally 500μl PBS were added and, again, the tubes were capped and vortexedcontinually for about 2 minutes. The capped tubes were next centrifugedin a microfuge at high speed for 2 minutes to facilitate cleanseparation of the aqueous and organic solvent phases. The absorbance of280 run of the upper aqueous layer (1 ml) containing the GM-CSF wasdetermined and the concentration of GM-CSF in mg/ml was calculated bydividing the absorbance by the extinction coefficient of 1.08. Thesamples were then submitted for TF1 bioassay to determine thebioactivity of the GM-CSF.

FIG. 5 shows the percent bioactivity of the GM-CSF extracted from themicrospheres. The control is an aqueous GM-CSF sample that has gonethrough the same extraction process. These results indicate that theGM-CSF extracted from the microspheres has retained complete bioactivity8.

EXAMPLE 11

Preparation of PLGA Microspheres and PLGA Degradation Profiles.

Microspheres containing 100% Cytec, 0.7 dL/g intrinsic viscosity (I.V.)PLGA, 100% R104 PLA, and an 80:20 mixture of the PLGA:PLA were preparedby the silicone oil hardening process. Samples of each of thesemicrospheres were incubated at 37° C. in PBS for periods of 1, 2, 4, 6,8, 10, 14, 28, 42, and 56 days. Following incubation the microsphereswere dissolved in tetrahydrofuran (THF) making 1 to 2% solutions(microsphere wt/THF volume). The samples were then analyzed by gelpermeation chromatography (GPC) using Waters HPLC system with a styragelHR4E column (Waters) which was maintained at 30° C. throughout the GPCrun. ; Polystyrene narrow molecular weight range standards were used tocalibrate the column. Weight and number average molecular weights of thedegraded PLGA polymers were determined with Millenium GPC software(Waters). FIG. 6 below illustrates the weight average molecular weightsof each microsphere formulation as a function of incubation time. Notethat the degradation of microspheres prepared from the 100% Cytec 0.7I.V PLGA was incomplete over a 14 day incubation period whereas the80:20 mixture of Cytec 0.7 I.V./R104 was essentially degraded. In thisexample the R104 PLA acted as a degradation enhancer for themicrospheres.

Modifications and variations of the compositions and methods formanufacture and use described herein will be obvious to those skilled inthe art from the foregoing description. Such modifications andvariations are intended to come within the scope of the appended claims.

We claim:
 1. A method for delivering GM-CSF to a patient in need thereofcomprising administering to said patient a biocompatible controlledrelease formulation that comprises an effective amount of GM-CSFdispersed within a hydrogel comprising a synthetic biocompatiblepolymer, wherein said hydrogel absorbs water in an amount up to 90% ofthe final weight of the hydrated hydrogel; and further wherein saidpatient is selected from the group consisting of: a) a trauma victim; b)a patient undergoing surgery; c) a patient who is infected with HIV; d)a patient having a wound; e) a patient who has a severe infection; andf) a patient who is undergoing vaccination.
 2. The method of claim 1,wherein the hydrogel comprises a polymer that is bioadhesive.
 3. Themethod of claim 1, wherein the controlled release formulation furthercomprises a pharmaceutically acceptable carrier.
 4. The method of claim1, wherein the hydrogel comprises a biodegradable synthetic polymer thatis a chemoattractant for white blood cells.
 5. The method of claim 4,wherein the chemoattractant polymer is selected from the groupconsisting of polyhydroxy acids, polyanhydrides, poly(ortho esters) andpolyphosphazenes.
 6. The method of claim 1, wherein the controlledrelease formulation is administered by injection.
 7. The method of claim1, wherein the controlled release formulation is administered orally. 8.The method of claim 1, wherein the controlled release formulation isadministered topically.
 9. The method of claim 1, wherein the controlledrelease formulation further comprises a compound selected from the groupconsisting of a degradation enhancer, a stabilizer, a solubilizer, and abuffering agent.
 10. The method of claim 1, wherein the hydrogel isformed from a polymer selected from the group consisting of ionicallycrosslinkable polysaccharides, synthetic biodegradable polymers andproteins.
 11. The method of claim 10, wherein the polymer is selectedfrom the group consisting of alginate, polyphosphazenes, polyacrylates,polyethylene oxide-polypropylene glycol block copolymers and hyaluronicacid.
 12. The method of claim 1, wherein the hydrogel is complexed andstabilized with polyions.
 13. A method according to claim 1, wherein thehydrogel is formed into microparticles.
 14. A method according to claim1, wherein the patient is a trauma victim.
 15. A method according toclaim 1, wherein the patient is undergoing surgery.
 16. A methodaccording to claim 1, wherein the patient is infected with HIV.
 17. Amethod according to claim 1, wherein the patient has a wound.
 18. Amethod according to claim 1, wherein the patient is undergoingvaccination.
 19. A method according to claim 13, wherein the patient isa trauma victim.
 20. A method according to claim 13, wherein the patientis undergoing surgery.
 21. A method according to claim 13, wherein thepatient is infected with HIV.
 22. A method according to claim 13,wherein the patient has a wound.
 23. A method according to claim 13,wherein the patient is undergoing vaccination.
 24. A method according toclaim 13, wherein the patient has a severe infection.